The field of tissue engineering and regenerative medicine is making strides in succeeding the field of organ and tissue replacement for the improved safety and affordability of medical care and the increase of patient lifespan and improvement of quality of life. In the midst of advancement in the field, many hurdles remain in the path to implementation of the techniques developed for regenerating bone following massive tissue loss caused by injury or disease or regenerating other body tissues, e.g., skin. It is generally accepted that a compressive strength of 5 MPa and an elastic modulus of 50 MPa make a material suitable as a scaffold for bone regeneration [1-2].
Several polymers—both natural and synthetic—are of great interest in the tissue regeneration field and are being investigated for use as tissue engineering scaffolds [3]. This class of materials is so diverse and versatile that it can be made suitable for many applications. However, finding an ideal material has proven to be a challenge. The approach in many applications of engineered tissue is to introduce a 3D polymeric or ceramic scaffold seeded with cells into a defect site, where the scaffold provides structure, essential nutrient, and growth factors to the cells proliferating and differentiating in the defect site. [4]. While it provides a temporary template for the newly formed tissue, it is resorbed harmlessly by the body. When the scaffold is intended for bone regeneration, achieving the proper mechanical characteristics, namely the strength and stiffness of the support, becomes integral to its success [5].
One promising biopolymer is collagen, which is a key component of the extracellular matrix produced by differentiated osteoblasts during bone formation [3]. It has shown great potential for other tissue engineering applications, or in composites of other materials, but by itself lacks the compressive strength to be applied to bone regeneration [6]. Furthermore, at more than $150/g, collagen is quite costly and its use in a large implant would likely be prohibitively expensive for the average patient. Other popular natural scaffolds include fibrin and hyaluronic acid hydrogels [7], which possess similar limitations. In addition other additives have been proposed for scaffolds and other ways to generate the scaffold, including electrospinning [39-42].
Whey Protein
As an important staple in the food industry, whey protein and its components have been subjected to in-depth characterization and study, though primarily as they relate to food science and engineering [8-9]. In various journals information can be found concerning the rheological properties of whey protein solutions of less than 10% WP [10], the onset of gelation of protein solutions below 20% [11], and extensive information on properties such as flavor, foaming, texture, film properties, and the like [12]. Numerous studies have determined the correlation between protein concentration and mechanical and rheological properties of whey protein isolate gels using a variety of conditions, fabrication methods, and gel compositions. These studies have used protein concentrations equal to or lower than about 20% [27-29, 43, 44]. A micro-porous membrane was developed using an acidic mixture of whey protein isolate in concentrations from 30-40%, 0.015M-0.1M calcium chloride, and optionally a surfactant. The mixture was adjusted to pH 6.15 and centrifuged to remove the gases before heating to 120° C. on a baking sheet [21].
Bovine whey protein has been shown to promote the growth and differentiation of osteoblasts in different species [13-16] and to suppress osteoclast activity, preventing bone resorption [17]. Whey protein isolate is extremely inexpensive and abundantly available. Recent years have shown an increased drive to develop uses for whey protein in order to increase the value of milk products and reduce disposal costs and organic pollution [18-19]. Whey is considered a byproduct in cheese production, and the cheese manufacture industry pays for its disposal, as whey constitutes 80-90% of the original milk volume [20]. One study investigated the use of whey protein gels as non-fouling filtration membranes [21].
The components of the WPI protein mixture are well characterized [9] both in structure and in sequence [22-23], and its gelling properties have been extensively studied and are favorable for the application. Information on whey protein solutions and gels at low concentrations is known primarily as it relates to food science [24-29]. Whey protein is heat sensitive so thermal denaturing can be done at low temperatures, making thermal curing of protein solutions straight-forward. Added calcium ions participate in cross-linking, hydrogen bonding, and hydrophobic interactions on cooling, thus tightening the network and forming a strong matrix [8, 37-38].
Bovine whey protein has been shown to promote the growth and differentiation of osteoblasts across species [14-16], while suppressing osteoclast activity [17]. The role of osteoblasts is to construct and remodel bone tissue, while osteoclasts dissolve bone minerals and break down bone. The immunogenicity of WPI using WPI biofilms (10% WPI with glycerol or diethylene glycol) has been found to be benign in mice when implanted for up to 60 days [30].
Calcium chloride is added to improve gelling properties [31, 37, 43]. In an extensive study covering different salts and their relative impacts on the viscosity and gelation ability of whey protein solutions, calcium chloride ranked among the best gel-inducing salts [32]. These results have since been reproduced in other studies [11, 21], making the precursor suspension similar to the well-studied solutions of lower protein content.
Nanocomposites have been shown to drastically enhance the mechanical properties of a polymer matrix [33]. Polysaccharides were selected due to the proven ability of cellulose to reinforce a polymer matrix [34-35], and because it has been suggested that their hydro lytic degradation products may serve as an added nutrient source for proliferating cells. A built-in nutrient source would improve the growth and mineralization characteristics and expand the feasible scaffold dimensions—generally physically limited by insufficient diffusion into the scaffold interior [36].
U.S. Pat. No. 6,337,198 discloses a biodegradable and biocompatible porous scaffold for tissue engineering, using several polymers including, for example, hydroxycarboxylic acid and copolymers thereof, bisphenol-A based polyphosphoesters, and tyrosine-derived diphenol compounds.
U.S. Pat. No. 6,753,004 discloses a biodegradable fishing lure formed from a material which includes sucrose, gelatin, sodium alginate, locust bean gum, calcium chloride, starch, corn syrup, glycerin, sodium benzoate, and sodium metaphosphate. Whey is listed as one potential protein component.
U.S. Pat. No. 7,556,800 discloses a fishing lure comprised of fibrous collagen.
U.S. Pat. No. 7,615,593 discloses hydrogels where a polymer matrix is modified to contain a bifunctional poly(alkylene glycol) molecule covalently bonded to the polymer matrix, including polymer matrix made from whey protein gels.
U.S. Patent Application Publication No. 2006/0008445 discloses a fishing lure comprised of a matrix of fibrous collagen.